Retroviral vectors are gene transfer vehicles for birds and mammals that exploit features of the retrovirus replication cycle such as high infection efficiency and stable co-linear integration of the virally transmitted information in a target cell chromosome. Retroviral vectors are becoming important tools in basic research, biotechnology and gene therapy.
Most retroviral vectors currently in use are derived from Murine Leukemia Viruses (MLVs). MLVs are particularly suitable as vectors due to their well-documented pattern of transcription in diverse cell types and relatively simple modular genetic structure.
1. Retroviral structure Retroviruses belong among the enveloped viruses (FIG. 1). The bilipid envelope is derived from the host cell membrane and modified by the insertion of the viral surface protein (SU) and transmembrane protein (TM). The matrix protein (MA) is situated just under the outer membrane surrounding the inner core. The core consists of capsid protein (CA). Inside the capsid are two copies of the retroviral genome which are attached to each other at the 5' end via hydrogen bonding. The virus core particle also contains the viral enzymes: reverse transcriptase (RT), protease (PR), and integrase (IN), and the nucleocapsid protein (NC) which is bound to the viral genome. Besides these proteins encoded by the virus, the virion also contains a number of tRNA molecules derived from the host cell tRNA population.
Murine leukemia viruses (MLV) belong to the simple retroviruses. Retroviruses have a characteristic genomic map: Two long terminal repeats (LTRs) flanking the three structural genes gag, pol and env (FIG. 2). The LTRs can be subdivided into three regions: The U3 region containing the enhancer and promoter elements recognized by the cellular transcription machinery, the R region which play an important role during reverse transcription and furthermore contains the polyadenylation signal, and the U5 region containing sequences of importance in reverse transcription aid packaging of the retroviral genome. Additionally, the LTRs contain cis elements, the inverted repeats, important during the process of integration (FIG. 3).
The integrated provirus gives rise to two mRNA transcripts, a full-length mRNA encoding the gag-, and the gag-pol poly-proteins, and a spliced mRNA encoding the envelope glycoproteins. The full-length mRNA also serves as the genomic RNA and, besides the already described components of the LTR moieties, contains three important cis elements in the 5' untranslated sequence. The primer binding site (PBS), situated downstream from the U5 region, consists of 18 nucleotides complementary to the 3' end of the primer tRNA molecule. Also located in the 5' untranslated region, between the PBS and the beginning of the gag open reading frame, is the packaging signal (.PSI.). The 5' untranslated region furthermore contains a dimer linkage domain responsible for the dimerization of the two viral genomes in the virion. Immediately upstream from the U3 region is another important cis-element, the polypurine tract (PP), which consists of a stretch of A and G residues. This element serves as a site for priming plus-strand DNA synthesis during reverse transcription.
2. The retroviral lifecycle
The retroviral lifecycle is outlined in FIG. 4. Two different mechanisms have been proposed to explain the entry of the virus particle into the host cytoplasm. Most retroviruses, including MLV, are thought to enter the host cell through receptor-mediated endocytosis, a process in which the whole enveloped virus particle is internalized into an endosomal body. The receptor molecule for the ecotropic murine leukemia viruses has been cloned and identified as a cationic amino acid transporter.
After the viral core particle has entered the cytoplasm of the host cell, all enzymatic functions leading to the integrated double-stranded DNA provirus are managed by viral proteins synthesized in the previous host cell and brought along in the virion. The fate of the viral proteins after entry of the core particle is not clear, but the reverse transcriptase, the integrase and the capsid protein remain with the RNA genome forming the nucleoprotein complex in which reverse transcription occurs. Recently, also the matrix protein has been found in association with the nucleoprotein complex.
Following reverse transcription, the nucleoprotein complex migrates to the host cell nucleus. The mechanism responsible for the nuclear localization is unclear, although evidence from Rous sarcoma virus (RSV) suggests the IN protein to be important since the RSV IN protein, when produced in mammalian cells, is localized in the nucleus. Entry of the nucleoprotein complex into the nucleus requires mitosis, probably because the nucleoprotein complex cannot penetrate the nuclear envelope. Once in the nucleus, integration is mediated by the IN protein. The IN protein recognizes the conserved inverted repeats at the ends of the LTRs and removes 2 bases from the 3' hydroxyl termini of both strands. The IN protein also catalyzes a cleavage in the host DNA and mediates the connections between the proviral DNA and the host DNA. As for the specificity of integration no consensus host DNA target sequence has been found, although a tendency to integrate near DNase I-hypersensitive sites has been reported.
For the simple retroviruses (including MLV) transcription and translation is performed by the host cell machinery. Complex viruses (including HIV and HTLV) encode transactivating proteins involved in transcriptional regulation. The assembly of MLV particles takes place at the host membrane, and the process coincides with the budding process. In mammalian B and C type viruses (MMTV and HTLV, respectively) viral core particles are assembled in the host cell cytoplasm. Encapsidation of the viral genomic RNA is mediated through binding of the cis-acting encapsidation signal and the NC moiety of the Gag- or the Gag-Pol precursor protein.
After budding, the Gag- and Gag-Pol polyproteins are cleaved by viral protease (PR). Maturation of the viral proteins results in an overall change in virion morphology. In addition to proteolytic cleavage of the viral polyproteins following budding, the genomic RNA also undergoes a maturation process resulting in a compact dimeric genome.
3. Reverse transcription
The enzyme reverse transcriptase was discovered in 1970, and the current model of reverse transcription was proposed in 1979 (Gilboa et al. 1979). In addition to a DNA polymerase that can copy either RNA or DNA templates, reverse transcriptase contains an RNase H which selectively degrades RNA in RNA/DNA hybrids.
Retroviruses utilize a cellular tRNA molecule as a primer during reverse transcription. Different retroviruses utilize different tRNA species as primers in reverse transcription. Murine leukemia viruses (MLVs) and human T-cell leukemia virus (HTLV) use a proline-tRNA primer, human immunodeficiency virus (HIV) and mouse mammary tumor virus (MMTV) utilize a lysine-tRNA primer, whereas avian leukosis sarcoma virus (ALSV) use a tryptophan-tRNA molecule as a primer for reverse transcription. However, within a given group of retroviruses, the sequence of the primer binding site is highly conserved during retroviral replication, where the 3' 18 nucleotides of the tRNA molecule are copied during plus-strand synthesis. Furthermore, during the viral life cycle the tRNA primer is likely to specifically interact with viral proteins and the viral genome during the processes of packaging, annealing, and reverse transcription. Specific interactions between the tRNA primer and the viral proteins reverse transcriptase and the nucleocapsid protein, as well a secondary interactions between the tRNA and viral genomic RNA have been demonstrated in several viruses.
In a recent article (Lund et al. 1993) the present inventors have altered the MLV wild-type PBS matching a proline-tRNA to sequences matching either a glutamine- or a lysine-tRNA. The effect of the altered PBS sequences was studied by single cycle replication of a retroviral vector, enabling them to quantify the effect of the introduced mutations. The structure of the transduced PBSs was analyzed by amplification of proviral vectors followed by direct sequencing of the PBS and surrounding DNA. They found that MLV can replicate by using various tRNA molecules as primers and concluded that primer binding site-tRNA primer interactions are of major importance for tRNA primer selection, but that efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site. Later they have proved that MLV can also replicate by using the methionine (initiator)-tRNA as the primer.
The tRNA primer is packaged in the virion during virus assembly, and originates therefore from the previous host cell. The tRNA primer molecule interacts through base pairing with a region of the genomic RNA (the primer binding site) situated downstream from the U5 region at the 5' end of the genome (FIG. 5). In murine leukemia viruses 18 bases from the aminoacceptor stem and the T.PSI.C loop of the tRNA are annealed to the PBS and the reverse transcription of the retroviral genome is initiated from the 3' end of the tRNA molecule. From here reverse transcriptase synthesizes the first DNA, the minus-strand strong stop DNA. The 3' end of the minus-strand strong stop DNA contains a copy of the R region complementary to the 3' R region of the genomic RNA. The newly synthesized DNA is thought to relocate to this region (first jump) from where minus-strand DNA synthesis can proceed. As the growing minus-strand is synthesized, RNase H continues to degrade the viral RNA. A fragment of the viral RNA, situated at the polypurine tract (PP), is left and acts as the primer for plus-strand DNA synthesis. Plus-strand DNA synthesis proceeds through U3, R and U5 and copies the bases of the tRNA molecule that are complementary to the PBS. At position 57 in the tRNA molecule the reverse transcriptase encounters a modified nucleotide (m.sup.1 A), which it presumably cannot use as a template, and synthesis of the plus-strand terminates. RNase H removes the overhanging part of the tRNA molecule. The remaining part of the plus-strand, leading to the complete double-stranded DNA provirus, is primed by the first part after a relocation event where the two complementary copies of the PBS interact through base pairing. Resulting from this complex reaction is a double-stranded DNA provirus, which is longer than the RNA template due to the copying of repetitive sequences U3 and U5.
4. Retroviral vector systems
A central element in this invention is a retroviral vector propagation system in which infectious, recombinant virus particles can be produced without contamination with replication competent wild type virus. The main advantage of retroviral vectors is the utilization of the efficient infection and integration processes developed in the viruses through evolution.
The proviral genome of MLV can be divided into sequences that are required in cis or in trans for viral replication (FIG. 6). The cis-acting elements are located at the ends of the proviral genome and encompass the U3, R and U5 regions, the inverted repeats in U3 and U5, the PBS, the polypurine tract and she packaging signal. These sequences contain all the elements needed for correct reverse transcription and integration and make up the minimal requirements for a retroviral vector. The space needed for the insertion of foreign DNA is created by deleting the sequences encompassing the open reading frames gag, pol and env.
For most applications it is essential that the retroviral vectors used for gene transfer are unable to generate new virus progeny in the target cell. This requires that the vector constructs are replication defective, the components required to complete the life cycle being supplied from loci outside the vector construct in the virus producing cell.
If these components are not present in the target cell, further virus generation is not possible. However, replication competent viruses may arise as a result of recombination. Such virus may eventually have pathological consequences including the malignant transformation of the infected target cell.
Retroviral vectors are normally propagated by the use of specialized packaging cell lines. In such packaging cell lines all trans-acting virus-encoded components are produced from loci outside the vector construct.
The first packaging cell line was constructed by Mann et al. (1983). This cell line (.psi.-2), containing a Moloney-MLV proviral genome with a 350 bp deletion overlapping the packaging signal, produces all the viral proteins needed in trans for virion production (FIG. 7A). After transfection into this cell line, retroviral vectors carrying the necessary cis-signals will be packaged into infectious virions (FIG. 7B). Although the early packaging cell lines .psi.-2 (Mann et al. 1983) and .psi.-AM.sup.1 (Cone & Mulligan 1984) have been used successfully in a large number of studies, they suffer from important deficiencies. Despite the deletion of the packaging signal replication competent viruses may arise, presumably as a result of recombination between the packaging construct and introduced vector sequences or viruses endogenous to the genome of the packaging cell (Miller 1990). Recombination occurs at high frequency in retroviral replication. The rate of homologous recombination between two heterologous genomes packaged in the same virion has been estimated to range as high as 10 to 30% for each round of replication. A number of studies have reported recombination between introduced viruses or vectors and homologous sequences endogenous to the host cell. Further evidence that deletion of the packaging signal is not sufficient to prevent packaging, comes from a study of a retroviral vector without a packaging signal where packaging and transfer of this vector was easily detected, even though the transduction rate was 3000 fold lower than that of a vector containing the packaging signal. Obviously, the 350 bp deletion in the 5' untranslated region does not inhibit packaging, a fact supported by reports indicating that sequences within the gag open reading frame and within the U5 region also may play a role in the encapsidation process. Furthermore, retroviral particles also package non-viral RNA, indicating that both specific recognition and a more general affinity for RNA is involved in the encapsidation process. Moreover, only a single recombination event is needed to restore the packaging signal and regenerate replication competent viruses. There are several reports of helper virus regeneration resulting from homologous recombination between the packaging construct and either introduced vectors or endogenous viral sequences. FNT .sup.1 The .psi.-AM packaging construct resembles .psi.-2 but expresses another env gene giving rise to recombinant viruses with an amphotropic host range.
To further reduce the risk of generation of complete viruses by recombination in the packaging cells, second generation packaging cell lines have been constructed. The main advantage of these new packaging cell lines is that the packaging construct has been divided into two separate constructs; one encoding the gag-pol polyprotein and one carrying the env gene (FIG. 8). By splitting the packaging construct the risk of recombination is greatly reduced (Miller 1990). By reducing the sequence homology of the packaging construct to the introduced vectors and endogenous MLV-like sequences the risk of homologous recombination can be further reduced.
However, not only the engineered parts of the packaging cells but also endogenous retroviral sequences in the DNA of the packaging cell as well as in the target cell may have to be considered in safety assessments. The risk of a contribution from endogenous retroviruses may be reduced by use of packaging cells based upon cell lines from other species where the endogenous retroviral elements may be more divergent. The current packaging cell technology has recently been reviewed (D. Valerio 1992).
A recent publication (Chapman et al. 1992) describes that the yeast retrotransposon Tyl uses tRNA.sub.i.sup.Met as a primer for Ty1 reverse transcription, and that mutations in the Ty1 element that alter 5 of the 10 nucleotides that are complementary to the tRNA.sub.i.sup.Met abolish Ty1 transposition. When a yeast strain is constructed which lacks wild-type tRNA.sub.i.sup.Met and is dependent on a mutant derivative of tRNA.sub.i.sup.Met that has an altered acceptor stem sequence, engineered to restore homology with the Ty1-PBS mutant, the compensatory mutations made in the tRNA.sub.i.sup.Met alleviate the transposition defect of the Ty1-PBS mutant. The mutant and wild-type tRNA.sub.i.sup.met are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1-PBS, and from this the authors conclude that complementarity between the Ty1-PBS and tRNA.sub.i.sup.met is essential for transposition, but is not necessary for packaging of the tRNA inside virus-like particles.